Circ_0043947 contributes to interleukin 1β-induced injury in chondrocytes by sponging miR-671-5p to up-regulate RTN3 expression in osteoarthritis pathology

Objective Osteoarthritis (OA) is a chronic joint disease featured by articular cartilage degeneration and damage. Accumulating evidence have demonstrated the pivotal regulatory roles of circular RNAs in OA pathology. However, the role of circ_0043947 in OA progression and its associated mechanism remain largely unknown. Methods The expression of RNA and protein was determined by reverse transcription-quantitative polymerase chain reaction and Western blot assay. Cell viability was assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cell proliferation was analyzed by 5-Ethynyl-2′-deoxyuridine (EdU) assay and flow cytometry. Cell apoptosis was assessed by flow cytometry. Enzyme linked immunosorbent assay was conducted to analyze the release of pro-inflammatory cytokines. Dual-luciferase reporter assay and RNA immunoprecipitation assay were performed to confirm the target interaction between microRNA-671-5p (miR-671-5p) and circ_0043947 or reticulon 3 (RTN3). Results Interleukin 1β (IL-1β) stimulation up-regulated the expression of circ_0043947 in chondrocytes. IL-1β treatment restrained the viability and proliferation and induced the apoptosis, extracellular matrix degradation and inflammatory response of chondrocytes partly by up-regulating circ_0043947. Circ_0043947 interacted with miR-671-5p, and miR-671-5p silencing largely reversed circ_0043947 knockdown-mediated protective effects in IL-1β-induced chondrocytes. miR-671-5p interacted with the 3′ untranslated region (3′UTR) of RTN3. miR-671-5p overexpression attenuated IL-1β-induced injury in chondrocytes, and these protective effects were largely overturned by the overexpression of RTN3. Circ_0043947 acted as a molecular sponge for miR-671-5p to up-regulate RTN3 level in chondrocytes. Conclusion Circ_0043947 silencing alleviated IL-1β-induced injury in chondrocytes by targeting miR-671-5p/RTN3 axis.


Introduction
Osteoarthritis (OA) is a common degenerative joint disease involving the disease processes of all joint tissues, including cartilage and meniscal degeneration, subchondral bone remodeling, synovial inflammation and inflammation and fibrosis of the infrapatellar fat pad [1][2][3][4].  17:177 Several factors, including genetics, ageing, repetitive joint injury, obesity, metabolic disorders and anatomical factors related to joint shape and alignment, may initiate OA pathology [5,6]. In recent years, the central role of inflammation in OA has been gradually discovered [7]. Interleukin-1β (IL-1β) is a vital pro-inflammatory factor, which promotes the release of matrix metalloproteinases (MMPs) into the articular cavity and induces cartilage degradation [8]. Therefore, inhibition of these inflammatory mediators may be a potential therapeutic method for OA patients. Circular RNAs (circRNAs) are characterized by stable circular structure that formed by back-splicing at the 5′ and 3′ terminals [9]. Recently, accumulating articles have pointed out the vital functions of circRNAs in the pathology of human diseases due to their regulatory roles in gene expression [10,11]. Previous study has shown that multiple circRNAs are dysregulated in OA tissues compared with normal cartilage [12]. Wu et al. demonstrated that circ_0005105 contributes to extracellular matrix (ECM) degradation through mediating microRNA-26a (miR-26a)/NAMPT axis in OA [13]. In this study, we evaluated the role and working mechanism of a novel circRNA, circ_0043947, in OA pathology using IL-1βinduced chondrocytes.
It is generally accepted that circRNAs can serve as competing endogenous RNAs (ceRNAs) to competitively bind to miRNAs, thereby leading to the up-regulation of the downstream genes [14]. Increasing studies have associated dysregulated miRNAs with multiple pathological changes in OA, including articular cartilage destruction and chondrocyte apoptosis [15,16]. starbase and circinteractome bioinformatics databases predicted the potential interaction between circ_0043947 and miR-671-5p. Previous studies reported that miR-671 suppresses OA development and attenuates IL-1β-induced damage in chondrocytes [17,18]. In this study, we tested the target relationship between circ_0043947 and miR-671-5p and further explored their functional relevance in OA progression.
miRNAs can induce the degradation or translational repression of mRNAs by interacting with their 3′ untranslated region (3′UTR) [19]. Through starbase bioinformatics database, we found that the 3′UTR of reticulon 3 (RTN3) was complementary with miR-671-5p. Fu et al. demonstrated that LINC02288 promotes the apoptosis and inflammatory response of chondrocytes partly by up-regulating RTN3 via absorbing miR-374a-3p [20]. In this study, we analyzed the target relationship between RTN3 and miR-671-5p and further explored their functional association in OA pathology.
In this study, we explored the important molecules and signal pathways involved in OA pathology using IL-1β-induced chondrocytes as cell model. The role of circ_0043947 in regulating the phenotypes of IL-1βinduced chondrocytes was initially analyzed. Then, the downstream mechanism of circ_0043947 was explored by bioinformatics analysis and functional experiments.

RNase R and Actinomycin D (Act D) treatment
The circular structure and stability of circ_0043947 were tested using exonuclease RNase R and transcriptional inhibitor Act D, respectively.
RNA samples were treated with RNase R (100 μg/mL; Applied Biological Materials, Vancouver, Canada) for 20 min. Then, RT-qPCR analysis was performed to determine RNA levels. Chondrocytes were incubated with Act D (2 mg/mL; Sigma, St. Louis, MO, USA) for 4 h, 8 h or 12 h, and RT-qPCR was conducted to analyze RNA levels.

IL-1β stimulation
Chondrocytes were seeded onto 6-well plates at the density of 2.0 × 10 5 cells/well. Chondrocytes were stimulated with 10 ng/mL IL-1β (PeproTech, Suzhou, China) for 24 h. The treated chondrocytes were utilized for further analysis.

Reverse transcription-quantitative polymerase chain reaction (RT-qPCR)
Chondrocytes were seeded onto 6-well plates at the density of 2.0 × 10 5 cells/well. After the indicated treatment, RNA samples were isolated with Trizol reagent (Thermo Fisher Scientific, Foster City, CA, USA). The reverse transcription was conducted using a commercial RevertAid First-Strand complementary DNA (cDNA) Synthesis kit (Thermo Fisher Scientific) and stem-loop reverse-transcription universal primer (Ribobio, Guangzhou, China). cDNA was amplified using RT-qPCR mix (Thermo Fisher Scientific) and specific primers ( Table 1). The fold changes were assessed by the 2 (-Delta Delta C(T)) method [21] with the references of glyceraldehydephosphate dehydrogenase (GAPDH; for circRNAs and mRNAs) and U6 (for miRNAs).

Flow cytometry
Flow cytometry was conducted to analyze cell cycle progression and apoptosis of chondrocytes.
Chondrocytes were seeded onto 6-well plates at the density of 2.0 × 10 5 cells/well. Chondrocytes were processed using Muse Cell Cycle Assay Kit (Millipore, Billerica, MA, USA), and the percentages of chondrocytes in G0/G1, S, and G2/M phases were assessed using flow cytometer (BD Biosciences, San Jose, CA, USA).
Chondrocytes were seeded onto 6-well plates at the density of 2.0 × 10 5 cells/well. Chondrocytes were processed using commercial cell apoptosis kit (BD Biosciences). Chondrocytes were simultaneously stained with fluorescein isothiocyanate (FITC)-conjugated Annexin V and propidium iodide (PI) at room temperature for 15 min in the dark, and the percentage of chondrocytes with FITC + and PI ± was analyzed using flow cytometer (BD Biosciences).

Western blot assay
Chondrocytes were seeded onto 6-well plates at the density of 2.0 × 10 5 cells/well. After the indicated treatment, cells were disrupted with radio-immunoprecipitation assay (RIPA) buffer (Beyotime, Jiangsu, China), and protein samples were quantified using the commercial bicinchoninic acid assay kit (Beyotime, Shanghai, China). Protein samples (25 μg/lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the protein bands were then blotted onto the polyvinylidene difluoride (PVDF) membrane (Bio-Rad). The membrane was incubated with 5% skimmed milk for 1 h at room temperature to block the non-specific sites. The membrane was incubated with diluted primary antibodies (Table 2) overnight at 4 °C. Subsequently, the membrane was incubated with horseradish peroxidase (HRP)-labeled secondary antibody (dilution: 1:5000; Abcam, Cambridge, MA, USA) for 2 h at room temperature. Protein signals were appeared using the electrochemiluminescence detection system (Thermo Fisher Scientific). The intensities of protein bands were quantified by the Image Lab analysis software (Bio-Rad). Enzyme linked immunosorbent assay (ELISA) The concentrations of interleukin 6 (IL-6), IL-8 and tumor necrosis factor α (TNF-α) in the culture supernatant were assessed using the ELISA kit (Elabscience Biotechnology, Wuhan, China).

Dual-luciferase reporter assay
The linear sequence of circ_0043947 or the 3′ untranslated region (3′UTR) of RTN3, including the putative binding sites with miR-671-5p, was inserted into pGL3 plasmid (Promega, Fitchburg, WI, USA). The above sequences with mutant miR-671-5p binding sites were also cloned into pGL3 plasmid (Promega). The re-constructed plasmids were named as pGL3-circ_0043947 WT, pGL3-circ_0043947 MUT, pGL3-RTN3 3′UTR WT and pGL3-RTN3 3′UTR MUT. HEK293T cells were seeded onto 24-well plates at 5 × 10 4 cells/well and were co-transfected with 100 ng luciferase reporter plasmids and 50 nM oligonucleotides. After transfection for 48 h, the changes in luciferase activities were determined using the Dual Glo Luciferase Assay System (Promega). The luciferase activity of Firefly was normalized to Renilla luciferase activity.

RNA immunoprecipitation (RIP) assay
Chondrocytes were seeded onto 6-well plates at the density of 2.0 × 10 5 cells/well and were transfected with miR-NC or miR-671-5p. At 48 h after transfection, RIP assay was conducted using Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore). Chondrocytes were disrupted with RIP lysis buffer for 5 min on the ice. RNA samples were then incubated with antibody-coated magnetic beads. The antibody against Argonaute2 (AGO2) or Immunoglobulin G (IgG) was purchased from Abcam. RT-qPCR was implemented to analyze RNA enrichment.

Statistical analysis
The data were analyzed by GraphPad Prism 7.0 software (GraphPad, La Jolla, CA, USA) and were expressed as mean ± standard deviation (SD). The comparisons were analyzed using Student's t-test (in two groups) or oneway analysis of variance (ANOVA) followed by Tukey's test (in multiple groups). P < 0.05 was considered as statistically significant.

IL-1β up-regulates circ_0043947 expression in chondrocytes
Circ_0043947 (393 nt) was generated from the backsplicing of exon 18-23 of its host gene breast cancer 1, early onset (BRCA1) (Fig. 1A). RNase R digestion significantly reduced the level of BRCA1 mRNA, while the level of circ_0043947 remained almost unchanged in RNase R group and Mock group (Fig. 1B), suggesting that circ_0043947 was a circular transcript without 5′ or 3′ end. Subsequently, transcriptional inhibitor Act D was used to analyze the stability of circ_0043947 and BRCA1 mRNA. We found that circ_0043947 was more stable than its linear form BRCA1 (Fig. 1C). To explore the role of circ_0043947 in OA progression in vitro, we established OA cell model by exposing chondrocytes to IL-1β (10 ng/mL; 24 h). We found that IL-1β treatment markedly up-regulated the expression of circ_0043947 in chondrocytes (Fig. 1D). Overall, circ_0043947 might be implicated in the regulation of OA progression.

Circ_0043947 silencing protects chondrocytes against IL-1β-induced injury
Transfection with circ_0043947-si#1 or circ_0043947-si#2 markedly reduced circ_0043947 expression in chondrocytes, especially in circ_0043947-si#1 group ( Fig. 2A), thus we selected circ_0043947-si#1 for the subsequent loss-of-function experiments. IL-1β stimulation decreased the viability of chondrocytes, and cell viability was largely recovered by the silence of circ_0043947 (Fig. 2B). EdU assay and flow cytometry were conducted to analyze the proliferation ability of chondrocytes. We found that IL-1β suppressed the proliferation of chondrocytes, and the silence of circ_0043947 largely rescued the proliferation ability of chondrocytes ( Fig. 2C-E). IL-1β stimulation induced the apoptosis of chondrocytes, and cell apoptosis was notably suppressed by circ_0043947 interference (Fig. 2F). Western blot assay showed that circ_0043947 interference rescued the levels of proliferation-associated proteins (proliferating cell nuclear antigen (PCNA) and Cyclin D1) and reduced the level of pro-apoptotic cleaved caspase 3 (c-caspase 3) (Fig. 2G,  H), which further demonstrated that circ_0043947 interference promoted the proliferation and suppressed the apoptosis of IL-1β-treated chondrocytes. The main component of ECM (Collagen II) and two matrixdegrading enzymes (MMP3 and MMP13) were determined by Western blot assay. The results revealed that circ_0043947 interference reduced the levels of MMP3 and MMP13 whereas increased Collagen II expression in IL-1β-treated chondrocytes (Fig. 2I), indicating that circ_0043947 interference protected chondrocytes from IL-1β-induced ECM degradation. Cell inflammatory response was also assessed. IL-1β treatment promoted the production of inflammatory cytokines (IL-6, IL-8 and TNF-α), and cell inflammation was restrained by circ_0043947 interference (Fig. 2J-L). These results demonstrated that IL-1β-induced injury in chondrocytes was largely based on the up-regulation of circ_0043947.

Circ_0043947 silencing-mediated protective effects in IL-1β-induced chondrocytes are largely based on the up-regulation of miR-671-5p
RT-qPCR assay verified the high transfection efficiency of anti-miR-671-5p in chondrocytes (Fig. 4A). To investigate whether the function of circ_0043947 in IL-1βinduced chondrocytes was associated with miR-671-5p, we performed rescue experiments. Circ_0043947 silencing up-regulated miR-671-5p level, and the introduction of anti-miR-671-5p reduced its level again in IL-1βinduced chondrocytes (Fig. 4B). The addition of anti-miR-671-5p decreased cell viability again (Fig. 4C). As verified by EdU assay and flow cytometry, circ_0043947 knockdown-mediated protective effect on the proliferation of IL-1β-induced chondrocytes was largely reversed Fig. 3 Circ_0043947 acts as a molecular sponge for miR-671-5p. A The miRNA targets of circ_0043947 were predicted by bioinformatics algorithms using starbase and circinteractome databases. B The putative binding sites with circ_0043947 in miR-671-5p were shown. C Overexpression efficiency of miR-671-5p mimics in HEK293T cells was determined by RT-qPCR. D Dual-luciferase reporter assay was applied to verify the binding relation between circ_0043947 and miR-671-5p. E RT-qPCR assay was conducted to analyze the overexpression efficiency of miR-671-5p in chondrocytes. F RIP assay was performed to confirm the target relation between circ_0043947 and miR-671-5p. G The effect of circ_0043947 silencing on the expression of miR-671-5p was analyzed by RT-qPCR assay. H The expression of miR-671-5p was examined in IL-1β-induced chondrocytes (10 ng/mL, 24 h) by RT-qPCR assay. **P < 0.01, ***P < 0.001 by miR-671-5p silencing (Fig. 4D, E). miR-671-5p knockdown induced cell apoptosis again in circ_0043947silenced chondrocytes upon IL-1β exposure (Fig. 4F). Consistent with above results, Western blot assay uncovered that circ_0043947 silencing-mediated effects on the proliferation and apoptosis of IL-1β-induced chondrocytes were largely overturned by the interference of miR-671-5p (Fig. 4G). miR-671-5p knockdown up-regulated the expression of matrix-degrading enzymes (MMP3 and MMP13) and reduced the level of Collagen II (Fig. 4H), suggesting that miR-671-5p silencing induced ECM degradation in circ_0043947-silenced chondrocytes upon IL-1β exposure. Circ_0043947 knockdown suppressed IL-1β-induced inflammation, and cell inflammation was triggered again by miR-671-5p silencing (Fig. 4I-K). These results suggested that circ_0043947 silencing protected chondrocytes against IL-1β-induced effects partly by up-regulating miR-671-5p.

RTN3 overexpression overturns miR-671-5p overexpression-induced effects in IL-1β-induced chondrocytes
miR-671-5p overexpression reduced RTN3 protein level, and RTN3 protein expression was largely rescued with the addition of RTN3 plasmid in IL-1β-induced A The predicted binding sequence with miR-671-5p in RTN3 was predicted by starbase database. B Dual-luciferase reporter assay was conducted to validate the target interaction between miR-671-5p and RTN3. C and D The effect of miR-671-5p overexpression or knockdown on the expression of RTN3 protein was determined by Western blot assay. E The protein level of RTN3 was measured in chondrocytes treated with IL-1β (10 ng/mL, 24 h) by Western blot assay. F Chondrocytes were transfected with circ_0043947-si#1 alone or together with anti-miR-671-5p, and the protein level of RTN3 was measured by Western blot assay. G The overexpression efficiency of RTN3 plasmid in chondrocytes was determined by Western blot assay. H Chondrocytes were transfected with miR-671-5p alone or together with RTN3 plasmid, and the protein expression of RTN3 was measured by Western blot assay. ***P < 0.001  (Fig. 6A). miR-671-5p overexpression rescued cell viability and proliferation and suppressed apoptosis, ECM degradation and inflammation in IL-1β-induced chondrocytes (Fig. 6B-J). The addition of RTN3 plasmid restrained the viability and proliferation of miR-671-5p-overexpressed chondrocytes upon IL-1β stimulation (Fig. 6B-D). Cell apoptosis was promoted in RTN3 and miR-671-5p co-transfected group in IL-1β-induced chondrocytes (Fig. 6E). Western blot assay showed that miR-671-5p overexpression protected chondrocytes against IL-1β-induced effects in the proliferation and apoptosis of chondrocytes partly through down-regulating RTN3 (Fig. 6F). RTN3 overexpression increased the levels of MMP3 and MMP13 and reduced the level of Collagen II in miR-671-5p-overexpressed chondrocytes upon IL-1β stimulation (Fig. 6G). Cell inflammatory response was also induced again in RTN3 and miR-671-5p co-transfected group in IL-1β-induced chondrocytes (Fig. 6H-J). These results suggested that miR-671-5p overexpression protected chondrocytes from IL-1β-induced injury partly by down-regulating RTN3.

Discussion
OA is a chronic degenerative disease induced by proinflammatory cytokines such as IL-1β [8]. Many researchers have associated the biological functions of circRNAs with OA pathology [22][23][24]. For instance, circ-SERPINE2 is reported to restrain OA progression by regulating miR-1271 and ETS-associated molecules [25]. Circ-IQGAP1 silencing is reported to attenuate IL-1β-mediated OA development by targeting miR-671-5p/TCF4 axis [26]. We found that IL-1β stimulation significantly up-regulated circ_0043947 expression in chondrocytes. IL-1β stimulation suppressed the viability and proliferation Fig. 6 RTN3 overexpression overturns miR-671-5p overexpression-induced effects in IL-1β-induced chondrocytes. A-J Chondrocytes were treated with IL-1β, IL-1β + miR-671-5p or IL-1β + miR-671-5p + RTN3 and their corresponding controls. A Western blot assay was implemented to analyze the protein expression of RTN3 in chondrocytes. B and C Cell proliferation ability was assessed by MTT assay and EdU assay. D and E Flow cytometry was utilized to analyze cell cycle progression and cell apoptosis. F and G The protein levels of PCNA, Cyclin D1, c-caspase 3, MMP3, MMP13 and Collagen II were measured by Western blot assay. H-J Cell inflammatory response was analyzed by ELISA. *P < 0.05, **P < 0.01, ***P < 0.001